The long-term goal of this project is to understand the molecular basis of inherited metabolic diseases caused by inborn errors of macromolecular catalytic machines. The model system under study is a family of highly conserved human alpha-ketoacid dehydrogenase complexes ranging from 4- to 10-million daltons in size, which comprises the branched-chain alpha-ketoacid dehydrogenase complex (BCKDC), the pyruvate dehydrogenase complex (PDC) and the alpha-ketoglutarate dehydrogenase complex (KGDC). These mitochondrial macromolecular structures catalyze the oxidative decarboxylation of alpha-ketoacids in the branched-chain amino acid and glucose degradative pathways. The organization of catalytic components is similar in that the 24- meric transacylase or the 60-meric transacetylase coupled with the E3-binding protein (in the case of human PDC) forms the structural core, to which multiple copies of a decarboxylase/dehydrogenase (E1), a dihydrolipoamide dehydrogenase (E3) are attached by non-covalent interactions. BCKDC is deficient in Maple Syrup Urine Disease (MSUD) manifested by often-fatal alpha-ketoacidosis, encephalopathies and mental retardation. Deficiency in human PDC results in impaired glucose utilization and neonatal lactic acidemias, and is associated with type 2 diabetes. In this competing renewal application, we will continue to dissect the structure and function of BCKDC as well as the biochemical and structural basis for MSUD. However, the scope of the investigation will be extended to include the cognate components of human PDC and human KGDC. Specific Aims are: 1) To corroborate a novel molecular switch that coordinates the two sequential half-reactions (decarboxylation and reductive acylation) catalyzed by the E1b component of human BCKDC;2) To dissect the structural and biochemical basis for human dihydrolipoamide dehydrogenase (E3) deficiency, i.e. the E3-deficient form of MSUD, and to delineate structural determinants for E3 binding in human BCKDC and KGDC;and 3) To determine three-dimensional structure of the multimeric human PDC scaffold and to assess the role of the E3-binding protein in the core assembly of human PDC. These studies are aimed to increase our understanding of how this family of large catalytic machines works at the biochemical and structural level, and how the conserved catalytic mechanisms and macromolecular assemblies are disrupted by disease-causing human mutations. The knowledge obtained from this investigation will provide a framework for developing more effective therapies for MSUD and neonatal lactic acidemias.